ABSTRACT The orchids Ophrys sphegodes and O. exaltata are reproductively isolated from each other by the attraction of two different, highly specific pollinator species. For pollinator attraction, flowers chemically mimic the pollinators' sex pheromones, the key components of which are alkenes with different double-bond positions. This study identifies genes likely involved in alkene biosynthesis, encoding stearoyl-acyl carrier protein (ACP) desaturase (SAD) homologs. The expression of two isoforms, SAD1 and SAD2, is flower-specific and broadly parallels alkene production during flower development. SAD2 shows a significant association with alkene production, and in vitro assays show that O. sphegodes SAD2 has activity both as an 18:0-ACP Δ(9) and a 16:0-ACP Δ(4) desaturase. Downstream metabolism of the SAD2 reaction products would give rise to alkenes with double-bonds at position 9 or position 12, matching double-bond positions observed in alkenes in the odor bouquet of O. sphegodes. SAD1 and SAD2 show evidence of purifying selection before, and positive or relaxed purifying selection after gene duplication. By contributing to the production of species-specific alkene bouquets, SAD2 is suggested to contribute to differential pollinator attraction and reproductive isolation among these species. Taken together, these data are consistent with the hypothesis that SAD2 is a florally expressed barrier gene of large phenotypic effect and, possibly, a genic target of pollinator-mediated selection.

[Show abstract][Hide abstract]ABSTRACT: Flax (Linum usitatissimum L.) is an important crop with many characteristic features such as its abundant essential ω-3 fatty acids for human nutrition. Fatty acid (FA) biosynthesis in plants, including flax, involves several consecutive steps governed by different gene families. Using in silico gene mining and comparative analysis, genome-wide gene identification and characterization were performed for six gene families related to FA biosynthesis, including KAS, SAD, FAD, KCS and FAT. We identified 91 FA-related genes from flax cv. CDC Bethune genome, from which seven previously cloned genes were validated. The newly identified 84 FA-related genes include 14 novel genes from the KAS family, two from the SAD family, 13 from the FAD2 family, three from the FAD3 family, 38 from the KCS family and 14 from the FAT family. Out of the 91 genes identified, 88 were duplicated as a consequence of recent whole genome duplication events, in which 13 FAD2 genes were hypothesized to have evolved from tandem gene duplication events followed by a whole genome duplication event and, more recently, by a single gene deletion. The six gene families described here are highly conserved in plants and have diverged anciently. These newly identified flax genes will be a useful resource for further research on FA gene cloning and expression, QTL identification, marker development and marker-assisted selection.

[Show abstract][Hide abstract]ABSTRACT: Episodes of rapid speciation provide unique insights into evolutionary processes underlying species radiations and patterns of biodiversity. Here we investigated the radiation of sexually deceptive bee orchids (Ophrys).Based on a time-calibrated phylogeny and by means of ancestral character reconstruction and divergence time estimation, we estimated the tempo and mode of this radiation within a state-dependent evolutionary framework.It appears that, in the Pleistocene, the evolution of Ophrys was marked by episodes of rapid diversification coinciding with shifts to different pollinator types: from wasps to Eucera bees to Andrena and other bees. An abrupt increase in net diversification rate was detected in three clades. Among these, two phylogenetically distant lineages switched from Eucera to Andrena and other bees in a parallel fashion and at about the same time in their evolutionary history.Lack of early radiation associated with the evolution of the key innovation of sexual deception suggests that Ophrys diversification was mainly driven by subsequent ecological opportunities provided by the exploitation of novel pollinator groups, encompassing many bee species slightly differing in their sex pheromone communication systems, and by spatiotemporal fluctuations in the pollinator mosaic.

[Show abstract][Hide abstract]ABSTRACT: Divergent selection by pollinators can bring about strong reproductive isolation via changes at few genes of large effect. This has recently been demonstrated in sexually deceptive orchids, where studies (1) quantified the strength of reproductive isolation in the field; (2) identified genes that appear to be causal for reproductive isolation; and (3) demonstrated selection by analysis of natural variation in gene sequence and expression. In a group of closely related Ophrys orchids, specific floral scent components, namely n-alkenes, are the key floral traits that control specific pollinator attraction by chemical mimicry of insect sex pheromones. The genetic basis of species-specific differences in alkene production mainly lies in two biosynthetic genes encoding stearoyl–acyl carrier protein desaturases (SAD) that are associated with floral scent variation and reproductive isolation between closely related species, and evolve under pollinator-mediated selection. However, the implications of this genetic architecture of key floral traits on the evolutionary processes of pollinator adaptation and speciation in this plant group remain unclear. Here, we expand on these recent findings to model scenarios of adaptive evolutionary change at SAD2 and SAD5, their effects on plant fitness (i.e., offspring number), and the dynamics of speciation. Our model suggests that the two-locus architecture of reproductive isolation allows for rapid sympatric speciation by pollinator shift; however, the likelihood of such pollinator-mediated speciation is asymmetric between the two orchid species O. sphegodes and O. exaltata due to different fitness effects of their predominant SAD2 and SAD5 alleles. Our study not only provides insight into pollinator adaptation and speciation mechanisms of sexually deceptive orchids but also demonstrates the power of applying a modeling approach to the study of pollinator-driven ecological speciation.

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Stearoyl-acyl carrier protein desaturases areassociated with floral isolation in sexuallydeceptive orchidsPhilipp M. Schlütera,b,1, Shuqing Xua,b,c, Valeria Gagliardinib, Edward Whittled, John Shanklind, Ueli Grossniklausb,and Florian P. SchiestlaInstitutes ofaSystematic Botany andbPlant Biology, University of Zürich and Zürich-Basel Plant Science Center, CH-8008 Zurich, Switzerland;cInstitute ofIntegrative Biology, Swiss Federal Institute of Technology Zürich and Zürich-Basel Plant Science Center, CH-8092 Zurich, Switzerland; anddDepartmentof Biology, Brookhaven National Laboratory, Upton, NY 11973Edited* by Wendell L. Roelofs, Cornell University, Geneva, NY, and approved February 23, 2011 (received for review September 6, 2010)The orchids Ophrys sphegodes and O. exaltata are reproductivelyisolated from each other by the attraction of two different, highlyspecific pollinator species. For pollinator attraction, flowers chemi-cally mimic the pollinators’ sex pheromones, the key componentsof which are alkenes with different double-bond positions. Thisstudy identifies genes likely involved in alkene biosynthesis, encod-ing stearoyl-acyl carrier protein (ACP) desaturase (SAD) homologs.The expression of two isoforms, SAD1 and SAD2, is flower-specificand broadly parallels alkene production during flower develop-ment. SAD2 shows a significant association with alkene production,and in vitro assays show that O. sphegodes SAD2 has activity bothas an 18:0-ACP Δ9and a 16:0-ACP Δ4desaturase. Downstream me-tabolism of the SAD2 reaction products would give rise to alkeneswith double-bonds at position 9 or position 12, matching double-bondpositionsobservedinalkenesintheodorbouquetofO.spheg-odes. SAD1 and SAD2 show evidence of purifying selection before,and positive or relaxed purifying selection after gene duplication.By contributing to the production of species-specific alkene bou-quets, SAD2 is suggested to contribute to differential pollinatorattraction and reproductive isolation among these species. Takentogether, these data are consistent with the hypothesis that SAD2is a florally expressed barrier gene of large phenotypic effect and,possibly, a genic target of pollinator-mediated selection.acyl-acyl carrier protein desaturase|isolation genes|pollination|speciationRspeciation. This statement is especially true for ecological specia-tion, in which divergent selection pressures on key traits drive theestablishment of reproductive isolation even in the absence ofgeographic barriers to gene flow (1). This process fits the genicview of speciation, in which only few loci of large effect may beresponsible for species differentiation, whereas gene flow is possi-ble throughout the rest of the genome (2, 3). In practice, thechallenge in studying these processes is identifying the traits underdivergentselectionandtheir genetic basis (1).Inplantswithstrongpollinator-mediated reproductive isolation (floral isolation), how-ever, key floral traits are direct targets of selection (1, 4). By iden-tifying the molecular mechanisms underlying these traits, genesdirectly involved in reproductive isolation (so-called “barrier” or“isolation” genes) or even speciation can be identified (3–5).Strong floral isolation and high pollinator specificity make sex-ually deceptive orchids an excellent system for identifying barriergenes (4, 6). Rewardless orchids of the genus Ophrys attract maleinsects by sexual mimicry, inducing mating attempts of pollinatorswith flowers, whereby pollen is transferred. The key component tothis system is the chemical mimicry of the pollinator female’ssex pheromone (7, 8), a blend of substances consisting mostly ofcuticular hydrocarbons, e.g., alkanes and alkenes. Alkenes (un-saturated hydrocarbons) areof special importance,and a differenteproductive isolation is a central topic in the study of evolu-tion,itsoriginandmaintenancebeingcriticalfortheprocessofproportion of alkenes was found to be the major odor differenceamong two closely related Ophrys species attracting differentpollinators (9). In Ophrys, speciation by pollinator shift has beenhypothesized, and there is evidence both for pollinator-drivengeneticdifferentiationandselectiononfloralhydrocarbonprofiles(4, 6, 9, 10). In particular, specific pollinators mediate strong floralisolation among the coflowering closely related species O. spheg-odes and O. exaltata by effectively preventing gene flow, whereasother reproductive barriers are largely absent (11). These speciesdiffer mainly in the double-bond position of their major alkenes(9), implying that the genes underlying this alkene difference maybe barrier genes (6).Although alkanes are common components of the wax layercovering the aerial parts of plants (12), alkenes are rare. Alkanesare synthesized from fatty acyl-coenzyme A (CoA) intermediatesthat undergo several rounds of chain elongation from the carboxylterminus. These fatty acid (FA) intermediates undergo reductionto aldehydes and decarbonylation to form alkanes, mostly pro-ducing odd-numbered alkanes from even-numbered very-long-chain fatty acid (VLCFA) intermediates (12, 13). Alkenes arethought to follow the same synthesis scheme, except for the in-troduction of double-bonds in an additional desaturation step (6).Notably, biosynthesis of the alkenes in insect sex pheromones islikely very different from that in plants. Although insect acyl-CoAdesaturases (which introduce the double-bond into alkene pre-cursors) were identified as putative speciation genes (3, 5), plantacyl-acyl carrier protein (ACP) desaturases that are responsiblefor the conversion of saturated to unsaturated FAs are mostlyunrelated to their animal counterparts (14). Specifically, planthomologs of the animal integral membrane acyl-CoA desaturasesactmostly on acyl-lipid intermediates.In contrast, soluble,plastid-localizedstearoyl-ACPdesaturases(SAD)carryouttheubiquitousdesaturationof18:0(saturatedC18)to18:1(monounsaturatedC18)FAintermediates(14).†SuchSADsarecandidatesfortheinsertionof a double-bond into the precursors of alkenes in plants (6, 10).Double-bond insertion at position Δ9of 18:0-ACP’s carbon chain(counting from the substituted end) by a Δ9-SAD would yieldAuthor contributions: P.M.S., J.S., U.G., and F.P.S. designed research; P.M.S., S.X., V.G.,E.W., J.S., and F.P.S. performed research; P.M.S., S.X., and V.G. analyzed data; and P.M.S.wrote the paper.The authors declare no conflict of interest.*This Direct Submission article had a prearranged editor.Data deposition: The sequences reported in this paper have been deposited in the Gen-Bank database (accession nos. FR688105–FR688110).1To whom correspondence should be addressed: E-mail: philipp.schlueter@systbot.uzh.ch.This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1013313108/-/DCSupplemental.†Shorthand notation for fatty acids and their derivatives is given in C:D form where Cspecifies the number of carbon atoms and D the number of double-bonds; the position xof a cis double-bond in the carbon chain is indicated by Δxsubstituted end (if applicable), or by ω-x when counting from the unsubstituted end.when counted from the5696–5701| PNAS| April 5, 2011| vol. 108| no. 14www.pnas.org/cgi/doi/10.1073/pnas.1013313108

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18:1Δ9-ACP. This product could be elongated, to e.g., 28:1Δ19-CoA (double-bond at position Δ19= ω-9, with ω counting fromthe unsubstituted end), leading to the production of 27:1Δ9alkenes upon decarbonylation. Therefore, species differences inalkene composition might result from changes in gene expressionand/or enzyme activity of specific SAD-encoding genes amongspecies, implying that such genes are candidate barrier genes inOphrys orchids.Here, we report the isolation of SAD homologs from O. spheg-odes and O. exaltata and discuss their potential role as barriergenes. Specifically, we address the following questions: (i) arethere any differences among species regarding SAD gene expres-sion or protein structure, (ii) are such differences associated withalkene production, (iii) are SAD proteins functional desaturases,and (iv) is there any evidence for selection on these enzymes?ResultsGene Cloning of Ophrys Stearoyl-ACP Desaturase Homologs. PutativeSAD-encoding transcripts were cloned by homology to Arabi-dopsis thaliana SSI2 (SUPPRESSOR OF SA-INSENSITIVITY2;At2g43710), the main Δ9-SAD-encoding gene of Arabidopsis.Three putative homologs, named SAD1–SAD3 (Fig. S1A), wereidentified from cDNA of Ophrys flower labella and their fullcoding sequence was obtained by RACE. SAD1 was identifiedonly from O. sphegodes, whereas the SAD2 and SAD3 genes werecloned from both species. SAD3 showed only silent substitutionsbetween the O. sphegodes and O. exaltata alleles (hereafter,denotedbyOsandOeprefixes).IncontrasttoSAD3,OsSAD2andOeSAD2 differed at the amino acid level (Fig. S1A).Evolutionary Analysis. Homologs of A. thaliana SSI2 (Table S1)wereidentifiedinpublicsequencedatabasesandusedtoconstructa Bayesian inference phylogeny of plant acyl-ACP desaturases(Fig. 1 and Fig. S2A). There was only one group of monocotdesaturases, with Ophrys SAD1 and SAD2 occupying a positionseparate from SAD3. This finding indicated that the gene dupli-cation events associated with plant desaturase diversification oc-curred after the split of monocots and eudicots. Furthermore, theSAD1/SAD2 dichotomy is more recent than the split of proto-SAD1/2 and SAD3. To test for the signature of selection, a maxi-mum likelihood-based analysis of synonymous mutations (dS;preserving the amino acid sequence) versus nonsynonymousmutations (dN; altering the amino acid sequence) was performed.This analysis revealed no indication of selection for SAD3.However, significant purifying selection (P = 0.002) was found onthe SAD1/SAD2 clade before the split of SAD1 and SAD2, andsignificant positive or relaxed purifying selection (all P < 0.001)thereafter (Fig. 1, Fig. S2, and Tables S2–S4). A more conserva-tive exact test of synonymous and nonsynonymous sites is con-sistent with this interpretation (Table S4).Cuticular Hydrocarbons and Gene Expression. Because high levels ofalkenes were found on flowers, but not on leaves of Ophrys (7),the occurrence of hydrocarbons and SAD expression in differentO.sphegodesandO.exaltatatissuesandfloraldevelopmentalstageswasinvestigated(Fig.2,Fig.S3,andFig.S4).Matureflowersofthetwo species differed significantly in the levels of different alkenes,withO.sphegodesproducinghighlevelsof9-alkenesand12-alkenes(strictlyspeaking,11/12-alkenes;seeMethods),andhighlevelsof7-alkenes in O. exaltata (Fig. S3 B and C). Expression of SAD1 andSAD2 (but not SAD3) differed among mature labella from thetwospecies (Fig. 2A). Together with the finding that SAD3 wasexpressed in leaf tissue lacking alkenes, this suggests that SAD1and/or SAD2 are involved in species-specific differences in alkeneproduction. While alkanes were found in all tissues, most alkeneswere barely detectable in leaves/bracts, sepals/petals, and labellafrom the smallest buds. The relative amount of alkenes, however,increased throughout flower development (Fig. 2B and Fig. S3 F–O).Asjudgedbysemiquantitativereversetranscriptase(RT)-PCR,SAD1,andSAD2expressionbroadlyparalleledalkeneoccurrence,but only SAD2 expression could significantly explain the presenceofseveral9-and12-alkenes,whicharedifferentamongspeciesanddetectable by pollinators (Fig. 2C and Fig. S3). Although SAD3showed a significant association with one species-specific 9-alkene(C25;Fig.S3B),itslackofspecies-specificexpressionpatternmakesit unlikely to be a causative factor.Protein Predictions.Becausecommonstearoyl-ACPdesaturasesareplastid localized (14), we checked whether a plastid transit peptidewas predicted for Ophrys SAD proteins. For SAD1 and SAD3 (butnot SAD2), the presence of a transit peptide was predicted (TableS5). However, moderate prediction scores and N-terminal se-quence divergence from the well-characterized Ricinus communisplant SAD (RcSAD) indicated that care is needed when postu-lating the subcellular localization of the Ophrys SADs. Using acrystal structure of RcSAD as a template, structural homologymodels were generated for OsSAD1, OsSAD2, OeSAD2, andOsSAD3 (which is identical in sequence to OeSAD3). Thesemodels were in good overall agreement, with differences amongprotein backbones localized mainly to one loop region (Fig. S1B).Geometry around the active site and substrate-binding pocketappeared to be mostly conserved among Ricinus and Ophrys pro-teins, and a canonical stearic acid (18:0) substrate modeled intoRcSAD fitted into Ophrys structures similarly well (Fig. S1C). ThemostprominentdifferencebetweenRcSADandOsSAD2isLeu123atthealiphaticendofthehypotheticalsubstrate-bindingcavity(Fig.S1C).BetweenOphrysSAD1andSAD2s,therewereseveralaminoacidchanges,mainlyontheproteinsurface(Fig.S1D),andtherewasa marked difference in isoelectric point (Table S5). Hypotheticallysubstrate-interacting regions were mostly similar among OsSAD2and OeSAD2, but OsSAD1 showed some amino acid differences BFig. 1.Bayesian phylogeny with branch lengths from BaseML; numbers indicateposterior probabilities (where >0.5) next to branches. Selected branches fororchid desaturases are labeled, and the respective dN/dSratios (from CodeMLfree-ratio model) are indicated in Inset. An asterisk marks branches A, B, andC, for which dN/dSratios are significant (P < 0.01) among one- and two-ratiomodels (Tables S2–S3).Phylogenetic analysis of SAD homologs, showing monocot clade.Schlüter et al.PNAS| April 5, 2011| vol. 108| no. 14| 5697EVOLUTION

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near the aliphatic end of the substrate-binding cavity (Fig. S1E).Overall, homology models suggested Ophrys proteins to be func-tional desaturases, although differences among the proteins in-dicated they might not be functionally equivalent.SAD Functional Characterization. Protein function of putative Oph-rys desaturases OsSAD1, OsSAD2, and OeSAD2 was investigatedin transgenic Arabidopsis and by in vitro assays of enzyme activity.The Ophrys SAD coding sequences were heterologously expressedin Arabidopsis under the control of the Cauliflower mosaic virus35S RNA promoter. None of the transgenic plant lines com-plemented the dwarf phenotype of homozygous ssi2 mutants (SIMethods), indicating that orchid transgenes could not fully func-tionally replace the A. thaliana desaturase SSI2. However, thepresence of the OsSAD2 transgene was significantly associatedwith changes in unsaturated C18and C16FA levels in Arabidopsisleaf lipids, suggesting that OsSAD2 has enzymatic activity in Ara-bidopsis (Fig. S5).To uncover thespecificreaction catalyzed by each Ophrys SAD,recombinant proteinswere assayed fordesaturase activityinvitro,using acyl-ACP from regiospecifically deuterated fatty acids. ForOsSAD1, no product was detectable by gas chromatographycoupled to mass spectroscopy (GC/MS), and lack of solubleOeSAD2 expression precluded its analysis. However, desaturaseactivity was observed for OsSAD2. Consistent with the lack ofcomplementation of Arabidopsis ssi2 mutants, in vitro OsSAD2activity was low. This low activity may reflect a requirement forspecific ACP or ferredoxin proteins different from those presentin enzyme assays or in Arabidopsis (cf. refs. 15 and 16). OsSAD2was active both on 18:0 and 16:0 substrates, producing 18:1Δ9and16:1Δ4products, respectively, as confirmed by MS of fatty acidmethyl esters (FAMEs) of reaction products and their pyrrolidinederivatives (Fig. 3). Considering fatty acid elongation from thecarboxyl end, these desaturation products would be expected togive rise to 9-alkenes and 12-alkenes, respectively.DiscussionReproductive isolation between O. sphegodes and O. exaltatadepends on the attraction of two different, highly specific polli-nator species by chemical mimicry of their sex pheromones (11).This specificity is due to the presence of alkenes with differentdouble-bond positions (7–9). During development, these alkenesaccumulate in the labella of Ophrys flowers. This accumulation isinmarkedcontrasttotheubiquitouspresenceofalkanesonorchidsurfaces, suggesting that alkene production is tissue- and stage-specific. Among the three putative orchid desaturases, SAD3showed a relatively constant expression without obvious speciesdifference,consistentwithafunctionasahousekeepingdesaturaserather than a factor linked to alkene production. By contrast,SAD1 and SAD2 expression broadly paralleled alkene production,and SAD2 showed a significant association with 9- and 12-alkenelevels in O. sphegodes, supporting a functional link. SAD1 andSAD2 probably originated by gene duplication, forming a lineagedistinct from SAD3. Purifying selection before this duplicationevent suggests a conserved role of the ancestral protein. Thehigher rate of amino acid change after duplication may indicatea partialrelease fromfunctionalconstraints,although,considering

that alkenes are likely under divergent selection (9), it is alsopossible that selection drove the divergence of protein function.Taken together, these results implicate Ophrys SAD2 as a desa-turase-encodinggeneassociatedwiththebiosynthesisofalkenesinthe floral pseudopheromones.OsSAD2 is a functional desaturase capable of producing18:1Δ9(ω-9) and 16:1Δ4(ω-12) FA intermediates from which 9-alkenes and 12-alkenes could be synthesized (Fig. 4). However,because housekeeping desaturase activity should be ubiquitousand not restricted to alkene-producing tissues, other proteinsmust be involved to ensure that desaturation products enter theVLCFA elongation pathway in flowers. For example, changes inthe activities of acyl-ACP thioesterase or acyl-CoA synthetaseisoforms (12, 13) would be potential candidates. Several orchidgenera related to Ophrys produce low levels of alkenes, whichmight have served as a preadaptation for sexual deception inOphrys (17). If so, changes in the relevant proteins should bepresent in both Ophrys and related genera.Different Ophrys species produce different alkenes, and double-bond differences will ultimately be due to desaturation reactions.Although several different mechanisms could potentially explaindifferences in desaturation among species, it appears that thehigher expression of SAD2 in O. sphegodes contributes to higher9- and 12-alkene levels in this species. Because OeSAD2 hardlydiffers from OsSAD2 around the active site and putative sub-strate-binding pocket (Fig. S1E), it is likely that both enzymescatalyze the same reaction. There are, however, amino acidchanges on the surface of SAD2 (Fig. S1D), so that an additionalactivitychangeduetodifferentinteractionswithreactionpartners(e.g., specific ACP or ferredoxin isoforms) (15, 16) cannot beruled out. Such a change may explain why only OsSAD2 affectedunsaturated FA levels in transgenic Arabidopsis. SAD1 differsfromSAD2byboth changeson theproteinsurfaceandchangesinFig. 4.than in O. exaltata (red arrows), due to expression (and possibly functional) differences. SAD2 reaction products are elongated and converted to 9- and 12-alkenes, the levels of which are higher in O. sphegodes than in O. exaltata. The exact source of high levels of 7-alkenes in O. exaltata is unknown. Floralalkenes are detected by pollinators, with 9- and 12-alkenes functioning as attractants to the bee Andrena nigroaenea (the pollinator of O. sphegodes).Conversely, the bee Colletes cunicularius (the pollinator of O. exaltata) is attracted by 7-alkenes, whereas 9-alkenes reduce this attraction. Overall, differentalkene blends in the two species lead to differential pollinator attraction associated with reproductive isolation.Model summarizing SAD2 involvement in floral isolation among O. sphegodes and O. exaltata. SAD2 activity is higher in O. sphegodes (blue arrows)Schlüter et al.PNAS| April 5, 2011| vol. 108| no. 14| 5699EVOLUTION

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thesubstrate-binding pocket,especiallywherethealiphaticendofthe substrate is expected to bind. However, two lines of evidencesuggest that SAD1 is not a functional desaturase: First, SAD1expression was not significantly associated with alkene pro-duction. Second, no evidence of SAD1 activity was detected ineither in vitro assays or transgenic Arabidopsis.The species-specific alkene differences associated with SAD2arebiologicallyrelevant(Fig.4).Electrophysiologicalstudieswiththe two specific pollinators, the solitary bees Andrena nigroaenea(for O. sphegodes) and Colletes cunicularius (for O. exaltata),showed that both detect 9-alkenes (C23, C25, C27, C29) and some12-alkenes (Andrena: C27, C29; Colletes: C29) (7, 8). Moreover,O. sphegodes alkene blends induced mating behavior in A.nigroaenea(7),whereasO.exaltataalkeneblendscontaining7-and9-alkeneswerelesseffectivethanonly7-alkenesforC.cunicularius(8), indicating that 9-alkenes may inhibit mating behavior in thispollinator. Taken together, these observations suggest that thealkenes linked to SAD2 activity are directly involved in the spec-ificity of pollinator attraction and, thus, reproductive isolationamong the two orchid species.Inconclusion, ourdataareconsistent withtheproposalthattheSAD2 desaturase underlies the phenotypic difference in 9- and12-alkenes among O. sphegodes and O. exaltata and, thereby, con-tributes to differential pollinator attraction and reproductive iso-lation among these species. SAD2 therefore represents a barriergene of large phenotypic effect on pollinator attraction by or-chid flowers.MethodsPlant Material. Plants of O. sphegodes Miller and O. exaltata Tenore subsp.archipelagi (Gölz & Reinhard) Del Prete were grown in a greenhouse at theBotanic Garden of the University of Zürich. For developmental stage-specificanalysis of hydrocarbons and gene expression, inflorescences were taken onthe first day of anthesis of the first flower of a given plant, flowers and budswere dissected, and the first open flower was used as a reference point.Gene Cloning and Expression Analysis. Total RNA was extracted from flash-frozen orchid tissue by using TRIzol reagent (Invitrogen) according to themanufacturer’s instructions, followed by assessment of RNA quality andquantity by agarose gel electrophoresis and spectrophotometry using anND-1000 (NanoDrop Technologies). Where necessary, RNA was further puri-fiedbyLiClprecipitation(18).TotalRNAwastreatedwithDNaseI(Fermentas)and reverse-transcribed into cDNA by using RevertAid M-MuLV H−ReverseTranscriptase (Fermentas), an anchored oligo-dT primer, and the supplier’sprotocol. Locus-specific and/or semiquantitative PCR was carried out by usingRedTaq ReadyMix (Sigma), the supplier’s protocol scaled to 10–20 μL withcDNA from 1 ng/μL total RNA as a template. For primers and cycling con-ditions, see Table S6 and SI Methods. Initial amplification of orchid SADfragments used a nested degenerate primer approach. PCR products werecloned into pDRIVE (Qiagen), positive clones were identified, and they wereSanger-sequenced by using BigDye 3.1 and a 3130XL Genetic Analyzer (Ap-plied Biosystems), as recommended by the manufacturers. Full-length codingsequence was isolated as detailed in SI Methods, deposited in GenBank (ac-cession nos. FR688105–FR688110), and amplified essentially as before (butreactions also containing 0.015 units per μL Pfu DNA polymerase; Promega)with modified PCR primers (Table S6) to engineer flanking attB sequencesduring PCR, as recommended by Invitrogen. AttB-site containing PCR prod-ucts of OsSAD1, OsSAD2, and OeSAD2 were cloned into pDONR207 by BPrecombination (Invitrogen) to give pENTR207-SAD, followed by selection onLB agar containing 10 μg/mL gentamicin, plasmid isolation, and sequenceconfirmation as described before.GC and GC/MS Analyses. Cuticular hydrocarbons were extracted by washingplanttissuein0.5mLofn-hexanefor1min,adding100ngofn-octadecaneasaninternal standard. GC was carried out as described (9), except for the use of alower heating rate of 4 °C/min. Retention times were compared against thoseof synthetic hydrocarbon standards run with the same settings. The standardswere: C19and C21–C29 n-alkanes and odd-chain (Z)-7-C21–C25, (Z)-9-C21–C29,(Z)-11-C25–C29and(Z)-12-C25–C27n-alkenes.SeveralsampleswerereanalyzedonanAgilent 5975 GC/MS with the same oven and column settings. Discriminationof (Z)-11/12 alkenes is not possible with these parameters. However, double-bond positionshavepreviouslybeen determined: Both studyspecies contain 11-and 12-alkenes, with 12-alkenes as the predominant isomer (19, 20). FAMEsextracted from Arabidopsis lines were analyzed by GC/MS using the same set-tings. FAMEs from desaturase assays were analyzed as in ref. 21.Plant Expression of Desaturases and Biochemical Activity Assay. To create2×35S:SAD expression vectors, pENTR207-SAD entry clones were recombinedwith the binary plant expression vector pMDC32 (22) by LR recombination(Invitrogen) and selected on kanamycin. Plasmids were isolated, sequenced, andtransformed into Agrobacterium tumefaciens strain LBA4404, which was, inturn, usedto transform A. thaliana line SALK_036854 (23) by using the floral dipmethod(24).ThislinecarriesaT-DNAinsertioninSSI2,associatedwitharecessivedwarf phenotype (SI Methods). Transgenic Arabidopsis plants were selected onMS(25)agarcontaining0.05%PlantPreservativeMixture(PlantCellTechnology)and 25 μg/mL hygromycin. Selected independent transgenic lines in an ssi2/ssi2background were tested for complementation: 35S:OsSAD1 (n = 2), 35S:OsSAD2(n = 5), and 35S:OeSAD2 (n = 2). Transgene expression (Fig. S5B) and sequencewere confirmed by RT-PCR and Sanger sequencing as described above. FAMEswere prepared by BCl3/methanol extraction (26).Different constructs for protein expression in Escherichia coli were madeand evaluated as detailed in SI Methods. Expression clones containing N-terminally modified orchid desaturases in the pET9d (Novagen) expressionvector were chosen for functional analysis. In these clones, amino acids 2–5(ELHL) were deleted to remove part of the putative chloroplast transitpeptide. Proteins were purified and assayed as described (21), with minormodifications: only 7,7,8,8-2H4-16:0-ACP and 12,12-2H2-18:0-ACP substrateswere used in assays containing 100 μg of desaturase, incubated for 2 h at22 °C. FAMEs were suspended in 50 μL of hexane for GC/MS analysis.Bioinformatic and Statistical Analyses. Molecular mass and isoelectric point ofproteins were predicted by using the ExPASy Server (27) and the presence ofa chloroplast transit peptide predicted using the ChloroP 1.1 server (28).Homology modeling was performed by using the SWISS-MODEL server (29)and the 2.4-Å crystal structure 1OQ4 (chain A) (30) of RcSAD as a template.Validation and quality checking of the models were done by using theProSA-web server (31) and Procheck software (32).Homologs of the Arabidopsis SSI2 desaturase gene (Table S1) wereextracted from public sequence databases as detailed in SI Methods andaligned based on amino acid sequence by using PRANK 0.91 (33). Poorlyalignable regions were excluded from downstream analysis. The GTR+I+ΓnucleotidesubstitutionmodelwasselectedbyusingMrModeltest2.2(34)andphylogenetic analysis conducted in MrBayes 3.1.2 (35), discarding results be-fore apparent convergence of analysis chains (burn-in 1 million of 30 milliongenerations). Branch lengths of the resulting consensus tree were optimizedwith BaseML and used as input for CodeML, both part of the PAML 4.3 (36)package. Different models of sequence evolution were calculated withCodeML and compared by likelihood ratio testing. Fisher’s exact tests weredone on (non)synonymous site counts (37) by using CodeML output. Statis-tical analyses were performed in Microsoft Excel and R 2.11.0 (38).ACKNOWLEDGMENTS. We thank A. Bolaños, A. Boyko, S. Cozzolino,M. Curtis, S. Kessler, M. and S. Schauer, and H. Zheng for providing labora-tory materials, help, or source code, and M. Anisimova and M. and S. Schauerfor discussions and comments. This work was supported by Austrian ScienceFund Fellowship J2678-B16 (to P.M.S.), Swiss Federal Institute of TechnologyZürich Grant TH 02 06-2 (to F.P.S.), the University of Zürich (U.G. and F.P.S.),and the Office of Basic Energy Sciences of the US Department of Energy (J.S.and E.J.W.).1. Schluter D (2009) Evidence for ecological speciation and its alternative. Science 323:737–741.2. Lexer C, Widmer A (2008) The genic view of plant speciation: Recent progress andemerging questions. Philos Trans R Soc Lond B Biol Sci 363:3023–3036.3. Wu C-I, Ting C-T (2004) Genes and speciation. Nat Rev Genet 5:114–122.4. Schiestl FP, Schlüter PM (2009) Floral isolation, specialized pollination, and pollinatorbehavior in orchids. Annu Rev Entomol 54:425–446.5. Noor MAF, Feder JL (2006) Speciation genetics: Evolving approaches. Nat Rev Genet 7:851–861.6. Schlüter PM, Schiestl FP (2008) Molecular mechanisms of floral mimicry in orchids.Trends Plant Sci 13:228–235.7. Schiestl FP, et al. (2000) Sex pheromone mimicry in the early spider orchid (Ophryssphegodes): Patterns of hydrocarbons as the key mechanism for pollination by sexualdeception. J Comp Physiol A Neuroethol Sens Neural Behav Physiol 186:567–574.5700| www.pnas.org/cgi/doi/10.1073/pnas.1013313108Schlüter et al.